Participation of protease‐activated receptor‐1 in thrombin‐induced microglial activation

Abstract

Activation of microglia, the resident macrophages in the CNS, plays a significant role in neuronal death or degeneration in a broad spectrum of CNS disorders. Recent studies indicate that nanomolar concentrations of the serine protease, thrombin, can activate microglia in culture. However, in contrast to other neural cells responsive to thrombin, the participation of novel protease-activated receptors (PARs), such as the prototypic thrombin receptor PAR1, in thrombin-induced microglial activation was cast in doubt. In this report, by utilizing primary microglial cultures from PAR1 knockout (PAR1-/-) mice, application of the PAR1 active peptide TRAP-6 (SFLLRN) in comparison to a scrambled peptide (LFLNR), we have unambiguously demonstrated that murine microglia constitutively express PAR1 mRNA that is translated into fully functional protein. Activation of the microglial PAR1 induces a rapid cytosolic free [Ca 2+ ] i increase and transient activation of both p38 and p44/42 mitogen-activated protein kinases. Moreover, although in part, this PAR1 activation directly contributes to thrombin-induced microglial proliferation. Furthermore, although not directly inducing tumor necrosis factor-a (TNF-a) release, PAR1 activation up-regulates microglial CD40 expression and potentiates CD40 ligand-induced TNF-a production, thus indirectly contributing to microglial activation. Taken together, these results demonstrate an essential role of PAR1 in thrombin-induced microglial activation. In addition, strategies aimed at blocking thrombin signaling through PAR1 may be therapeutically valuable for diseases associated with cerebral vascular damage and significant inflammation with microglial activation.

Lipopolysaccharide is a frequent and significant contaminant in microglia‐activating factors

“…In the case of pb-thr, there is strong evidence for the combination (Hanisch et al, 2004;Weinstein et al, 2005). Microglia are known to express PARs (Balcaitis et al, 2003;Suo et al, 2002Suo et al, ,2003 and as seen here (see Fig. 1) and previously (Weinstein et al, 2005), pharmaceutical-grade rh-thr induces small increases in CD95(Fas) and CD40 expression in the N9 cell line.…”

“…Dosages were selected to be within the range used in previously reported results (Badie et al, 2000;Hooper et al, 2005;Jarvis et al, 1999;Ryu et al, 2000;Suo et al, 2002;Weinstein et al, 2005). CD95 (Fas) a member of the TNF receptor family is a key microglial surface antigen and is upregulated in a variety of neuroinflammatory conditions (Choi and Benveniste, 2004;Lee et al, 2000).…”

Learning and memory deficits in mice lacking protease activated receptor-1

“…Notably, in adult animals, PAR1 is expressed at moderate to high levels in brain regions involved with emotional learning, including hippocampus and amygdala (Striggow et al, 2001). PAR1 has been extensively studied for its role in coagulation and hemostasis (Coughlin, 2000;Macfarlane et al, 2001), as well as in the survival of neurons following ischemic, traumatic, or neurotoxic insults (Gingrich & Traynelis, 2000a;Shibata et al, 2001;Riewald et al, 2002;Suo et al, 2002;Cheng et al, 2003;Junge et al, 2003;Xi et al, 2003;Guo et al, 2004;Olson et al, 2004;Hamill et al, 2005;Nicole et al, 2005). Surprisingly little, however, is known about PAR1's roles in normal brain function.…”

Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease

“…Thrombin-mediated microglial activation has been reported (Moller et al, 2000;Suo et al, 2002). Thrombin is generated from the precursor prothrombin that is endogenously expressed in human, mouse, and rat brain, including dopaminergic neurons in the SN (Dihanich et al, 1991;Soifer et al, 1994;Weinstein et al, 1995).…”

“…Thrombin is generated from the precursor prothrombin that is endogenously expressed in human, mouse, and rat brain, including dopaminergic neurons in the SN (Dihanich et al, 1991;Soifer et al, 1994;Weinstein et al, 1995). Thrombin-induced microglial activation involves protease-activated receptor-1 (PAR-1) (Suo et al, 2002). Recent studies demonstrated that direct injection of thrombin into various brain parenchyma including hippocampus and substantia nigra results in induction of iNOS, COX-2 and NADPH oxidase-mediated superoxide generation from microglial and subsequent neuronal degeneration (Choi et al, 2003;.…”